Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. Despite standard of care, withdrawal of heparin and use of a direct thrombin inhibitor (DTI), patients remain at significant risk for thrombosis. The pathogenesis of HIT has two major components: 1) formation of ultralarge complexes (ULC) of platelet factor 4 (PF4)/heparin (H)-containing immune complexes (IC) that activate platelets and other blood and vascular cells, and 2) events downstream of cellular activation by HIT IC, notably thrombin generation and action. DTIs do not prevent IC formation nor do they inhibit activation of platelets by HIT ICs. We hypothesize that combination therapy that targets critical effector molecules specific to HIT will be more effective than existing approaches to treatment. We recently reported that a specific inhibitor of Syk kinase, which mediates platelet activation via Fc?Rlla, prevents HIT-related thrombocytopenia and thrombosis in vivo in the HIT mouse model. We have observed that the Syk inhibitor is effective in reversing established HIT in vivo. In other preliminary studies, we identified novel small molecule PF4 antagonists (PAs) that prevent formation of PF4/H ULC immune complexes. We will pursue our studies in the following aims: Specific Aim 1: Characterize Syk inhibitors in depth as treatment of established HIT in model mice in vivo. Using the passive murine HIT system established by my group involving the infusion of a HlT-like monoclonal antibody KKO, we will assess the efficacy of the novel Syk inhibitor PRT318 to improve platelet counts and prevent new thrombosis. A novel systemic thrombosis imaging model, an arterial injury model, and a microvascular injury model will be used to best model the diversity of human HIT. These studies will also be extended to infusion of isolated clinical HIT IgGs to assess the clinical efficacy of PRT318. The safety of PRT318 will also be tested by measuring blood loss. Specific Aim 2: To identify PF4 antagonists with potential as HIT therapy. We will measure the ability of PAs to inhibit and reverse PF4/H ULC formation in vitro. We will quantify the potency of PAs to inhibit PF4/H-dependent platelet activation ex vivo, and perform proof of concept in vivo experiments with the most potent PAs. Two PAs are in hand with activity in the micromolar range. Specific Aim 3: To determine the effects of combination therapy directed at independent, sequentially acting targets in established HIT. The goal is to identify the superior therapeutic regimen of greatest relevance to human HIT. The experimental design is to combine these with standard DTI therapy: 1) Syk inhibitor PRT318 and 2) PAs in HIT model mice in vivo. We will then determine 3) the utility of combining the two novel agents (PRT318 and PA) with and without standard-of-care DTI. In summary, we believe that these proposed studies of two novel therapeutic agents that affect a major component of established HIT, alone or in combination with DTI, will improve outcome in HIT. Successful completion of these aims will bring new therapeutic candidates to clinical trials.